Bodily states in mammals, including disease states, are at least in part directly affected by proteins. Those proteins, acting directly or through enzymatic functions, contribute in large proportion to many diseases in animals and humans. In the past, therapeutic treatment has focused on interactions with those proteins in an effort to moderate the disease. Attempts have also been made to moderate the actual production of such proteins by interaction with molecules that directly affect their synthesis, i.e., the DNA sequence that underlies the gene product. It is well known that by interfering with the production of the proteins, the effect of the therapeutic results can be maximized. Likewise, therapeutic approaches have been developed that interfere with gene expression, leading to undesired formations which need to be patrolled. There are numerous methods that have been formulated for inhibiting specific gene expression which have been adopted to some degree and have been defined as antisense nucleic acids. The basic approach is that an oligonucleotide analog complimentary to a specific targeted messenger RNA or mRNA sequence is used. Pertinent references include those of Stein and Cohen (1988); Walder (1988); Marcus-Sekura (1988); Zon (1988); Van der Krol (1988) and Matteucci and Wagner (1996). Each of the foregoing concern general antisense theory and prior techniques.
Background of Antisense Technology
The term "antisense" generally connotes an approach to chemotherapy which is based upon the complementary pairing of an antisense oligonucleotide, or ASO, with a target nucleic acid. The use of an ASO compound requires a complementarity of the antisense base sequence to a target zone of an mRNA, so that the ASO will bind to that mRNA target sequence and will bring about selective inhibition of gene expression (D. A. Melton, 1988; Stein and Cohen, 1988; and Toulme and HelIIne, 1988). A more thorough understanding of such technology can be found in a book edited by Cohen (Oligonucleotides--Antisense Inhibitors of Gene Expression (1989)), and a recent review by Mercola and Cohen (1995).
Referring to FIG. 1, the schematic representation of the process of transfer of information from the DNA genome to a protein product can be seen. If the gene product (protein) is one whose controlled synthesis is essential for the well-being of the organism or cell, then a defective synthesis or an unwanted protein can lead to illness or death. FIG. 1 also illustrates that an antisense oligonucleotide (or oligo) can be used to inhibit or terminate protein synthesis.
A summary of the relevant portion of the disclosure from the review by Cohen (Id., 1989, pp. 1-6) is set forth below. There are five basic assumptions included in this approach:
(1) Cellular uptake: It is assumed that the oligo will cross the cell membrane and will be able to reach its target sequence within the cell. PA1 (2) Stability: The oligo will be stable under in vivo conditions, and will reach the target sequence in significant quantity. PA1 (3) Hybridization: The oligo will hybridize with the target sequence so that a DNA:RNA hybrid will be produced. PA1 (4) Inhibition of expression: The formation of this hybrid will prevent the expression of the gene(s) coded for by the hybridized mRNA. PA1 (5) Selectivity of binding: The oligo will not be bound non-selectively to many other sites, particularly protein sites so as to have its effective concentration, or potency reduced. PA1 N(1)+N(2)+N(3)+N(4) N(2)+N(6)+N(10)+N(14). PA1 N(5)+N(6)+N(7)+N(8)=N(1)+N(5)+N(9)+N(13). PA1 N(9)+N(10)+N(11)+N(12)=N(3)+N(7)+N(11)+N(15). PA1 N(13)+N(14)+N(15)+N(16)=N(4)+N(8)+N(12)+N(16). PA1 determining the fraction of each type of nearest-neighbor base pair in each target RNA:ASO-DNA hybrid sequence; PA1 substituting the nearest-neighbor base pair fraction into formulas to determine the fraction of each of the thirteen (13) polymers within contiguous segments of the target nucleic acid sequence; PA1 multiplying the fractions times the values of hybridization temperature (or thermodynamic energy) for the polymers to create a stability ranking and yield the relative hybridization temperatures for potential antisense oligonucleotides; and PA1 ranking the different nucleic acid segments for which relative hybridization strength has been determined according to hybridization potential, wherein the formulas used to identify the highest and lowest hybridization temperatures (or thermodynamic energies) along the length of the target nucleic acid sequence are a summation of the hybridization temperatures (or thermodynamic energies) as they relate to the relative base pairings. Alternatively, the nucleic acid segments can be ranked by other parameters, such as C+G content. PA1 providing a program and its identifier in a portion of a memory unit of the control computer; PA1 obtaining a nucleic acid sequence; obtaining a data list of values for combinations of nearest-neighbor nucleic acid pairs; PA1 obtaining a user-defined nucleic acid segment length value; PA1 determining the length of the DNA segment to be analyzed according to the nucleic acid segment length value used; and PA1 comparing the target nucleic acid sequence to the data list of values based on the user-defined nucleic acid segment length and calculating a nearest-neighbor value for each overlapping and contiguous length of the target nucleic acid sequence of said user-defined nucleic acid segment length to obtain a hybridization value for each segment having the user-defined length. The target sequence is analyzed in a concatenated fashion in all possible reading frames as shown for example in Table 1 below. PA1 a first set of binary code for obtaining a nucleic acid sequence in a first data format; PA1 a second set of binary code for comparing the nucleic acid sequence and the data list of values for combinations of nearest-neighbor nucleic acid pairs; and PA1 a third set of binary code for electronically routing the binary values to an output device. PA1 reading a nucleic acid sequence on a storage medium; PA1 obtaining a user-defined nucleic acid sequence segment length; PA1 separating the nucleic acid sequence into overlapping nucleic acid segments based on the user-defined nucleic acid sequence segment length; PA1 comparing the nucleic acid sequence that has been separated into nucleic acid segments to a data list of values for combinations of nearest-neighbor nucleic acid pairs; and PA1 determining a hybridization potential or ranking for each nucleic acid segment from the set of binary data. This computer-implemented process for determining the relative hybridization potential can be used to obtain a list that ranks hybridization potential based on the nearest-neighbor determination in a given, e.g. descending order.
The results of early studies that showed selective inhibition of gene expression in Rous sarcoma virus with a synthetic 13-mer (Zamenik and Stephenson, 1978) indicated that this oligo was indeed penetrating into cells. However, the general resistance to this possibility, and the difficulty in synthesizing oligos of such length until the mid-1980's, slowed progress in this area.
Once automated oligo synthesis became possible, further attempts were made to test this antisense approach. It has now been established that antisense oligonucleotides can be actively taken up by cells and can accumulate in the cellular nucleus (G. D. Gray et al., 1997).
Initial attempts to inhibit gene expression naturally concentrated on normal oligos with phosphodiester linkages. However, it has been believed that since these compounds are known to be subject to enzymatic hydrolysis by nucleases in vivo and in vitro, they could not form the basis of an effective antisense strategy. This problem of low oligo stability could explain the high concentrations of oligos that were found to be required to bring about inhibition of expression in some of the early work. Further, the breakdown of an informational molecule in which the integrity of the base sequence is integral to its mode of action would obviously be a devastating restriction on the strategy of the antisense approach.
Any chemical modification in an oligo can lead to a change in hybridization with its target mRNA sequence at physiological temperature. Since the object of this strategy is to ensure that hybridization occurs, it is mandatory that the hydrogen bonding capability of the bases not be impaired. It is for this reason that the modifications that are made in the oligo are usually in the backbone, and not in the bases or the sugars. Nevertheless, there are many possible modifications of the three portions of the nucleotide unit, only a few of which have so far been investigated. However, in order not to disrupt the formation of Watson-Crick base pairing, it is preferable that any modification that is made should be rather conservative. For example, the substitution of one sulfur atom for any oxygen or phosphate is perhaps the most conservative substitution that can be envisaged that accomplishes the aim of nuclease stability (Eckstein, 1985) without significantly impairing the hybridization of the oligo. The prospect for hybridization of an oligo with a mRNA must also take account of the fact that RNAs can have complex folded tertiary structures and, thus, the target sequence should be contained within a single-stranded accessible region.
The mechanics of inhibition of gene expression were originally presumed to arise from interference of the hybrid DNA:RNA duplex with ribosomal processing. This mechanism has been termed translation arrest or hybridization arrest. However, subsequent work has shown that ribonucleases that hydrolyze such hybrids, namely RNase-H, are actively involved in the mechanism of action (Walder and Walder, 1988). This was shown very clearly by comparison of normal oligos which have the beta configuration of the base-sugar linkage, and non-natural alpha-oligos, which form hybrids with RNA that are not susceptible to RNase-H, and which were concomitantly found not to produce translational arrest (Cavenave et al., 1989). The most potent antisense effects are obtained when RNA translation is blocked by RNase-H cleavage (Matteucci and Wagner, 1996).
It is important for the effectiveness of the antisense oligo approach that the oligo bind selectively to the target complementary sequence. If the oligo is bound non-selectively to the other sites, particularly protein sites that are present in the cell, the potency of any oligo as a putative drug would be severely limited. Eckstein has shown in a series of elegant studies (Eckstein, 1985) that phosphorothioate oligos have a tendency to bind to protein sites and to inhibit nucleases. This "problem" can, in fact, be turned into an advantage if the oligos interact specifically with a protein site, and thus inhibit, for example, a process that is required for vital proliferation. This has been found to be the case with the phosphorothioate analogues which inhibit certain polymerases in a sequence non-specific manner. Such an inhibition cannot be described as an antisense effect. However, the inhibition of HIV reverse transcriptase by phosphorothioate antisense oligomers (S-ASOs) (Matsukura et al.; 1987; 1988), and the effectiveness of ASOs have led to twelve clinical trials (Mercola and Cohen, 1995; Matteucci and Wagner, 1996) illustrating that phosphorothioates are particularly valuable. This is largely because phosphorothioates retain the ability to activate RNase-H.
If RNA is to be regarded as the legitimate target for this novel class of drugs, then much can be done to improve their effectiveness. One of the chief questions that arises is what part of the mRNA to target, for example one might target the 5' initiation codon as one of the preferred sites.
The covalent addition of intercalative or reactive groups onto either the 3' or 5' end of the oligo is already established as an appropriate modification of the antisense approach. In the former case, the intercalator is intended to enhance oligo binding, thus enabling short oligos to be used more effectively. While this would reduce the cost of an oligo, this tactic involves relatively non-selective interaction compared to a longer, more selective antisense base sequence. Alkylating agents attached to oligos have been used for chemical modifications of the other strand in a DNA duplex, but can also be adapted for DNA:RNA duplexes. The whole area of attaching hydrolyzing groups to the oligo (Stein and Cohen, 1988) or even ribonucleases (Corey and Schultz, 1987), is still very much in its infancy, although the cellular uptake and antigenicity of such putative oligo-enzymes is clearly a problem that must be addressed.
Referring to FIG. 2, a system for translation of mRNA is illustrated. More specifically, an mRNA polymer sequence 12 generally is acted on by ribosome machinery 10 such that if normal translation 14 occurs then nascent polypeptides 16 will readily be formed to give a sequence of amino acids. Conversely, if the mRNA binds an antisense DNA oligomer (ASO) sequence 18, a blocked translation 20 occurs, either because ribosomes 24 cannot translate or because RNase-H 26 cleaves at the hybrid sequence 22.
The antisense oligomer is generally DNA, instead of RNA, since RNase-H only attacks the RNA strand of DNA:RNA hybrids. Many chemical modifications of the antisense oligomer are possible, such as the substitution of phosphates in the sugar-phosphate linkages with phosphorothioate linkages. Set forth below is an illustration of the unmodified phosphate linkage and the phosphorothioate linkage. ##STR1##
Prior attempts to inhibit HIV gene synthesis by various antisense approaches have been made by a number of researchers. Zamecnik and coworkers have used a transcriptase primer site and splice donor/acceptor sites (P. C. Zamecnik et al., 1986). Goodchild and coworkers have made phosphodiester compounds targeted to the initiation sites for translation, the cap site, the polyadenylation signal, the 5' repeat region and a site between the gag and pol genes (J. Goodchild et al., 1988). In the Goodchild study, the greatest activity was achieved by targeting the polyadenylation signal. Agrawal and coworkers have extended the studies of Goodchild by using chemically modified oligonucleotide analogs, which were also targeted to the cap and splice donor/acceptor sites (S. Agrawal et al., 1988). Agrawal and coworkers have used oligonucleotide analogs targeted to the splice donor/acceptor site to inhibit HIV infection in early infected and chronically infected cells (S. Agrawal et al., 1989).
Sarin and coworkers have also used chemically modified oligonucleotide analogs targeted to the cap and splice donor/acceptor sites (P. S. Sarin et al., 1988). Zaia and coworkers, on the other hand, used an oligonucleotide analog targeted to a splice acceptor site to inhibit HIV (J. A. Zaia et al., 1988). Matsukura and coworkers synthesized oligonucleotide analogs targeted to the initiation of translation of the rev gene mRNA (M. Matsukura et al., 1987). Mori and coworkers used a different oligonucleotide analog targeted to the same region as targeted by Matsukura (K. Mori et al., 1989). Shibahara and coworkers used oligonucleotide analogs targeted to a splice acceptor site as well as to the reverse transcriptase primer binding site (S. Shibahara et al., 1989). Letsinger and coworkers synthesized and tested oligonucleotide analogs with conjugated cholesterol targeted to a splice site (Letsinger et al., 1989). Stevenson and Iversen conjugated polylysine to oligonucleotide analogs targeted to the splice donor and the 5' end of the first exon of the tat gene (Stevenson and Iversen, 1989). Buck and coworkers have recently described the use of phosphate-methylated DNA oligonucleotides targeted to HIV mRNA and DNA (H. M. Buck et al., 1990). In addition, a U.S. Patent issued to Ecker, has disclosed the use of synthetic oligonucleotides to inhibit the activity of the HIV virus (U.S. Pat. No. 5,166,195).
The need has, therefore, arisen for a systematic method and apparatus for identifying target DNA and RNA sequences in a predictable, logical pattern. Also required is a process that optimizes a ranking of nucleic acid sequences for targeting with antisense oligonucleotides. In addition, there is a long felt need for a means of assessing the thermodynamics and strength of nucleic acid hybridization in sequences that are to be the targets of antisense gene targeting technology in order to efficiently and expeditiously affect their expression. In Nature Biotechnology, Bernstein recently wrote, referring to antisense molecules, "the first step in their design--target identification--continues to be a major bottleneck" (Bernstein, 1998).